Western Blot- - Bands appear only nc eon the film! (Feb/19/2007 )

Hello All,

I have a peculiar problem..With one of my antibodies I notice that I can develop my film only once. (Its the same secondary antibody that I use for all my other blots.) Any attempts to get a second film draws a blank.I even tried immersing the membrane in fresh ECL solution but to no avail. Can somebody help me?

Thanks for your help!

-K

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How long do you expose your membran to the film? the signal gets weaker after some time.

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Hello K,

I was just wondering if you have solved this problem yet because I have the exact same problem occurring except I am using CPS which is even more sensitive than ECL! Aghh very frustrating! I was reading another post that suggested the secondary may be too concentrated for that particular antibody but am a little sceptical that this is the case for me but just to let you know incase this helps you. I have tried everything and so if you have been enlightened it sure would be great to know how. Good luck.

-smaher

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before you decide to be skeptical, please read this quote from Pierce:


"Signal Intensity and Duration
When all Western blotting factors are optimal, a
chemiluminescent signal can last for 6-24 hours,
depending on the specific substrate used. How
much light is generated and for how long depends
on the specific substrate being used and the
enzyme-to-substrate ratio present in the system.
Although the amount of substrate on a blot is
relatively constant, the amount of enzyme present
depends on how much was added and other
factors (Table 1). Too much enzyme conjugate
applied to a Western blot system is the single
greatest cause of signal variability, dark
background, short signal duration and low
sensitivity.
A signal emission curve that decays slowly
(Figure 1) is desirable as it demonstrates that each
component of the system has been optimized and
allows reproducible results. A signal that decays
too quickly can cause variability, low sensitivity
and lack of signal documentation. A long-lasting
signal minimizes variability with transfer
efficiency, different manufacturer lots of
substrate and other factors.

Although HRP continues its activity for as long
as substrate is available, a predictable statistical
probability exists for luminol to turn over HRP
and render it inactive. Free radical production
during the oxidation reaction can bind to HRP in
such a way that the enzyme can no longer interact
with the substrate. An abundance of HRP in the
system in turn produces an abundance of free
radicals that increases the probability of HRP
inactivation. Free radicals also can damage the
antigen, antibodies and the membrane,
prohibiting re-probing effectiveness

TRUST ME, A SIGNAL THAT BURNS OUT RAPIDLY CAN BE -AND OFTEN IS - DUE TO TOO MUCH SECONDARY ANTIBODY

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